The goal of this project will be to continue studies of the structure and function of lipid rafts: ordered sphingolipid and cholesterol-rich membrane domains found in mammalian and other eukaryotic cells. Among other functions, rafts are implicated in sorting of proteins and lipids between membranes, signal transduction, and some types of bacterial and viral infections. Prevoius studies in this project, as part of a (continuing) collaboration with the lab of Dr. Deborah Brown (Stony Brook), established some of the basic principles explaining how sphingolipids, sterols and certain proteins form rafts. New fluorescence and fluorescence quenching methods allowing detection of nanoscale rafts with full control over raft composition, combined with previously developed spectroscopic and biochemical techniques, will be used to define several of the still mysterious rules controlling lipid and protein participation in rafts. The basis of raft formation in the sphingolipid-poor plasma membrane inner leaflet will be studied in model membrane systems. Raft-forming behavior of biosynthetic precursors of cholesterol will be studied to help define how diseases blocking steps in cholesterol biosynthesis (e.g. Smith-Lemli-Opitz disease) may be related to deleterious changes in raft behavior. Ceramide displaces sterols from rafts. For this reason, biologically important ceramide-rich rafts will be studied in model systems and cells to define how they differ from ordinary rafts in terms of properties and protein interactions. To gain additional insights into the principles of raft formation, various small molecules with raft-promoting and raft-destabilizing behaviors will be studied in model membranes and cells. As part of these studies, the functional significance of the known raft-destabilizing effects of polyene antibiotics will be studied in both model membranes and cells. The interaction of proteins with rafts will be studied to define the relationship between protein structure and raft affinity. Transmembrane, lipid-anchored and cholesterol-binding proteins will be compared. Sterol analogs found to support raft formation to different degrees in the last grant period will be used to define the nature of protein sterol binding specificity. Whether proteins interact differently with ordinary and ceramide-rich rafts will also be determined. Finally, the degree to which proteins can regulate raft formation will be studied.